1. Field of the Invention
The invention relates to multiple-well plates, and in particular to high density plates for compound storage and biological assay.
2. Description of the Related Art
A plate is a container with multiple liquid reservoirs. It may have two to several thousand reservoirs (also called wells) depending on the application. The most common configurations have 96 or 384 wells. The Society for Biomolecular Screening sets the standard for plate geometry. Plates typically maintain a 127.76×85.47 mm footprint regardless of the number of wells. The number and spacing of the wells has been standardized around the 96-well plate which has 8×12 wells spaced 9 mm center-to-center. Other plates are based on this pattern. To increase the well density, multiply the number of wells in the x- and y-directions of a 96-well plate by an integer and divide the 96-well spacing by this same integer. For example, a 3456-well plate has six times the number of wells in both the x- and y-directions (or the orthogonal axes along which the wells are aligned), giving it 48×72 wells spaced at 1.5 mm.
FIGS. 1a-1b schematically illustrate a low density multiple-well plate 2 (FIG. 1a) in comparison with a high density multiple-well plate 4 (FIG. 1b). The low density plate illustrated at FIG. 1a includes multiple wells 6 spaced apart by approximately 9 mm in each direction or at least in the x-direction as shown. Each well 6 then occupies a 9 mm by 9 mm area of the plate. The density of wells 6 on the plate 2 is then one well per 81 mm2 or 0.012 wells/mm2. The total dimension of the plate 2 in the x direction is shown as 127.76 mm and that in the y direction is shown as 85.47 mm. Wells are absent only within an outer periphery of the plate 2, and so the area of active wells may be around approximately 8000 mm2. The total number of wells 6 that are fit onto the plate 2 is then the 8×12=96 wells 6 shown in FIG. 1a. 
The high density plate 4 illustrated at FIG. 1b includes multiple wells 8 in each 9 mm by 9 mm portion of the plate 4, instead of the single well shown in FIG. 1a. For example, each 9 mm by 9 mm area of the plate 4 of FIG. 1b may include 6×6=36 wells 8. The density of wells 8 in plate 4 of FIG. 1b is thus 36 times the density of the wells 6 of the plate 2 of FIG. 1a, or 36×0.012 wells/mm2 or 0.44 wells/mm2. The total dimension of the plate 4 in the x direction is shown as 127.76 mm and that in the y direction is shown as 85.47 mm, or the same as that of the plate 2 of FIG. 1a. So, the total number of wells 8 formed on plate 4 would be 96×36=3456 wells. It follows that the sizes of the wells 8 will be reduced compared to the sizes of the wells 4 approximately corresponding with the increase in well density. For example, the wells 8 would have a smaller diameter than the wells 4 by a factor of about six.
In recent years, the advantages of increasing the number of wells per plate have become apparent. We have seen manufacturers producing and the industry using plates with 864, 1536, 3456, and 9600 wells for example. FIG. 9 shows a table which sets forth the progression of plate well number as squared integer multiples of 96. The table also sets forth the evaporation barrier well numbers to arrive at the total microplate well number that includes the integrated evaporation barrier wells. (Microplate Well Number Table) The benefit of these high-density plates is twofold. First, more wells per plate mean fewer plates used. This is especially important in operations like high-throughput drug screening where hundreds of thousands of experiments are routinely executed in a day. Second, smaller wells mean less material used which is preferable because some reagents are very expensive or difficult to make.
The technical requirements for performing a large number of chemical or biological assays in parallel in such applications as high-throughput chemical compound screening have lead to the development of high-density multi-well plates in which a large number of miniaturized, identical wells are present on a single platform or plate. Types of platforms, multi-well plates, and accessory items such as plate lids and caddies or carriers are described in U.S. Pat. No. 6,426,050 to Coassin et al, which is herein incorporated in its entirety by reference. In general, the plurality of wells on a single plate enables the construction, for example, of an identical composition of assay reaction components in each well. Then a different single chemical compound is added to each well in order to screen a large number of chemical compounds for biological or chemical activity. Other applications include construction of different assay compositions in the different wells and then the addition of the same chemical compound to the different wells in order to screen for different biological or chemical activities of a single compound. To facilitate the development of assay construction and measurement instrumentation for the purpose of automating that instrumentation, an industry-standard format has been proposed (Astle, T., “Standards in robotics and instrumentation”, Journal of Biological Screening. Vol. 1, No. 4. pp. 163-169 (1996), which is herein incorporated by reference in its entirety) and maintained by the Microplate Standards Development Committee of the Society of Biomolecular Screening. The current revisions of this standard format, which comprise dimensional specifications for the footprint of the platform base, the height of the plate, and well dimensions and positions in the plate, provide a common set of useful definitions for the specification of multi-well platforms.
It is recognized in the present invention that it would be advantageous to make use of these definitions and standards for the positions of wells on the platform and for the height of the well bottom above the bottom of the supporting flange to enable compatibility with instrumentation configured for multi-well plates conforming to the proposed standard. In particular, it is further recognized that it would be advantageous to have a high-density planar array of wells in which the dimensions of the wells and their positions on the array are scaled according to the proposed standard. This would provide for ready modifications, typically in user-configurable software to enable compatibility with the wide range of automated instrumentation designed to be compliant with multi-well platforms manufactured to the proposed standard.
A number of multi-well platforms are commercially available for culturing cells, performing chemical or cellular assays, and for storing chemical compounds. Although many of these multi-well platforms offer necessary and desired features such as biocompatibility and low toxicity, substantial structural integrity and ease of manufacture, optical properties suitable for fluorescence and other spectrometric measurements, or chemical or thermal inertness, none of the present commercially available platforms offer all these desirable features combined into a single, multi-functional, low-cost plate. For example, Whatman Polyfiltronics offers a 96 well-format constructed of black polystyrene with a substantially optical-quality borosilicate Type II glass bottom that is suitable for fluorescence measurements due to the low intrinsic fluorescence of the bottom. The wall material of this plate, polystyrene, exhibits substantial autofluorescence at a wavelength of 460 nm when illuminated directly with light of 350 nm wavelength as taught by Coassin et al, U.S. Pat. No. 6,517,781, which is hereby incorporated in its entirety by reference. This intrinsic fluorescence of polystyrene adversely affects the sensitivity of a fluorescence assay when the well dimensions are decreased in a miniaturized, high-density format, because each well is supported by sufficient autofluorescent material to maintain the structural rigidity of the wall.
Adhesives used to bond glass and other transparent materials to the polystyrene plate bottom are soluble in ethanol and other solvents routinely used in chemical screening, thus limiting the functionality of the plate for storing concentrates of chemical compounds. The use of glass bottoms has proven optimal for spectrometric assays due to their high transmittance of light wavelengths most typically employed in chemical and biological assays (300 to 800 nm). Sealing the glass to the plastic to the interstitial material of the plate, however, may limit the use of the plate to particular reagents or solvents for reagents as well as the physical conditions such as temperature for storage or assay. One of the most common solvents used for storing chemical compound concentrates is dimethylsulfoxide (DMSO). Many adhesives and structural materials used in multi-well plate construction are not resistant to DMSO, so that plates used for chemical storage are typically constructed from materials such as polypropylene that are selected primarily on the basis of chemical resistance. These plate materials, although compatible with chemical storage, are typically not transparent and hence not useful for fluorescence assays.
Another problem is the potential chemical incompatibility of the material in an otherwise fluorescence-quality assay plate to the solvent such as DMSO used to maintain the chemical compound concentrate. The typical way this problem is addressed is by predilution of the concentrate from the storage plate into a diluent that is compatible with the assay plate, such as a buffer or other aqueous medium. But this requires the expenditure of an intermediate multi-well plate to perform the dilution, with its attendant compound management issue of keeping track of which wells receive which compound. Moreover, the attendant drawback of intermediate dilution is the decreased concentration of chemical compound that ultimately reached the assay. The intermediate dilution may be diluted further on the addition of other assay reagents or biological cells or other assay constituents that may be precluded from being present in the intermediate diluent. It is recognized in the present invention that it would be advantageous to have a system that overcomes these problems and difficulties by enabling the use of the same type of plate for both storing the compounds and assaying their activity.
It is further recognized in the present invention that it would be advantageous to provide a mechanically strong material for both the walls of the wells and the plate bottom. It is well appreciated by those skilled in the art that mechanical constraints are imposed when different materials are bonded together to achieve desired optical characteristics. For example, the differential thermal expansivities of glass and plastic result in their eventual detachment when subjected to repeated steam sterilization cycles to render a plate suitable for cell culture. Different materials are typically used in different parts of multi-well plates to achieve necessary mechanical or optical properties in those parts. The different abilities of these different materials to withstand mechanical stresses may however limit the usefulness of a particular plate designed and manufactured with optimization of only one property.
A difficulty encountered with small wells in high density plates is that many instruments designed to work with large wells in low-density plates no longer function properly when used to access small wells, largely because the instrument must generally be aligned more precisely with a small well than with a large one, all else being equal. Liquid handling instruments have other difficulties as well. First, for a pipette tip to fit into a small well, it must be thin, and thin pipette tips clog and break easily. Second, as the volume of liquid decreases, standard pipetting becomes less and less accurate and eventually fails altogether as surface tension becomes the dominant force.
Another issue that arises when dealing with small wells is evaporation. First is the difference in exposed liquid surface area between a large well and a small one. The ratio of surface area to volume in the well of a 3456-well plate is about four times that of a 96-well plate. For 3456-well plates and 96-well plates, this assumes well diameters of 1 mm and 7 mm with fill volumes of 2 μL and 200 μL respectively. Since evaporation rate is directly proportional to exposed surface area, a 1 mm diameter well would lose about 40% of its volume in the same time that a 7 mm well would lose 10%, assuming that all other conditions are the same. This brings us to a second evaporation issue that is recognized by the inventors in the present invention: all other conditions are generally not the same. In a lidded plate, small wells at the plate edges evaporate significantly faster than small wells at the interior of the same plate, which can be detrimental to an experiment being run or a chemical being stored in a plate.
In a plate with a lid, there is a small gap between the lid and the tops of the wells. In the interior of the plate, the liquid in the wells evaporates and becomes vapor, the partial pressure of that vapor increases in the space above the wells. This occurs until the system reaches equilibrium, at which point the liquid will cease to evaporate. The situation is different at the edges of the plate. Here the vapor being created diffuses away from the well and into the outside environment. The system does not reach equilibrium; instead, liquid continues to evaporate and vapor continues to diffuse away indefinitely. Small wells at the edge of the plate experience this phenomenon more drastically than large wells because their average distance from the edge is significantly shorter. A product referred to as a NanoWell Assay Plate manufactured by Aurora Biosciences Corporation has peripheral troughs designed to be filled with liquid to mitigate evaporation from wells at the edge of the plate. However, these troughs are difficult to fill, especially with automated equipment, and liquid in them tends to spill out easily. It is recognized in the present invention that it would be advantageous to have an improved high density, multiple-well plate that experiences reduced evaporation from peripheral wells.
Because high-density plates have so many wells, they lend themselves to applications where large numbers of different samples need to be interrogated. In high-throughput drug screening, for example, hundreds of thousands of distinct compounds are assayed for biological activity against a specific disease target. A typical pharmaceutical company will screen their compound library against perhaps hundreds of targets per year, generating tens of millions of data points. As mentioned, compounds are usually stored in 96- or 384-well plates and transferred into an assay plate with a pipetting device. Currently, only a small percentage of the pharmaceutical industry uses high-density plates for screening because of the difficulties mentioned above.